The flower industry strives to develop new and different varieties of flowering plants with improved characteristics ranging from disease and pathogen resistance to altered inflorence. Although classical breeding techniques have been used with some success this approach has been limited by the constraints of a particular species' gene pool. It is rare, for example, for a single species to have a full spectrum of coloured varieties. Accordingly, substantial effort has been directed towards attempting to generate transgenic plants exhibiting the desired characteristics. The development of blue varieties of the major cutflower species rose, carnation and chrysanthemum, for example, would offer a significant opportunity in both the cutflower and ornamental markets.
Flower colour is predominantly due to two types of pigment: flavonoids and carotenoids. Flavonoids contribute to a range of colours from yellow to red to blue. Carotenoids impart an orange or yellow tinge and are commonly the only pigment in yellow or orange flowers. The flavonoid molecules which make the major contribution to flower colour are the anthocyanins which are glycosylated derivatives of cyanidin, delphinidin, petunidin, peonidin, malvidin and pelargonidin, and are localised in the vacuole. The different anthocyanins can produce marked differences in colour. Flower colour is also influenced by co-pigmentation with colourless flavonoids, metal complexation, glycosylation, acylation, methylation and vacuolar pH (Forkmann, 1991).
The biosynthetic pathway for the flavonoid pigments (hereinafter referred to as the "flavonoid pathway") is well established and is shown in FIG. 1 (Ebel and Hahlbrock, 1988; Hahlbrock and Grisebach, 1979; Wiering and de Vlaming, 1984; Schram et al., 1984; Stafford, 1990). The first committed step in the pathway involves the condensation of three molecules of malonyl-CoA with one molecule of p-coumaroyl-CoA. This reaction is catalysed by the enzyme chalcone synthase (CHS). The product of this reaction, 2',4,4',6'-tetrahydroxychalcone, is normally rapidly isomerized to produce naringenin by the enzyme chalcone flavanone isomerase (CHI). Naringenin is subsequently hydroxylated at the 3 position of the central ring by flavanone 3-hydroxylase (F3H) to produce dihydrokaempferol (DHK).
The B-ring of dihydrokaempferol can be hydroxylated at either the 3', or both the 3' and 5' positions, to produce dihydroquercetin (DHQ) and dihydromyricetin (DHM), respectively. Two key enzymes involved in this pathway are flavonoid 3'-hydroxylase and flavonoid 3',5'-hydroxylase. The flavonoid 3'-hydroxylase acts on DHK to produce DHQ and on naringenin to produce eriodictyol. The flavonoid 3',5'-hydroxylase (hereinafter referred to as 3',5'-hydroxylase) is a broad spectrum enzyme catalyzing hydroxylation of naringenin and DHK in the 3' and 5' positions and of eriodictyol and DHQ in the 5' position (Stotz and Forkmann, 1982), in both instances producing pentahydroxyflavanone and DHM, respectively. The pattern of hydroxylation of the B-ring of anthocyanins plays a key role in determining petal colour.
Because of the aforesaid gene pool constraints, many of the major cutflower species lack the 3',5'-hydroxylase and consequently cannot display the range of colours that would otherwise be possible. This is particularly the case for roses, carnations and chrysanthemums, which constitute a major proportion of the world-wide cutflower market. There is a need, therefore, to modify plants and in particular roses, carnations and chrysanthemums, to generate transgenic plants which are capable of producing the 3',5'-hydroxylase thereby providing a means of modulating DHK metabolism, as well as the metabolism of other substrates such as DHQ, naringenin and eriodictyol. Such modulation influences the hydroxylation pattern of the anthocyanins and allows the production of anthocyanins derived from delphinidin, thereby modifying petal colour and allowing a single species to express a broader spectrum of flower colours. There is a particular need to generate transgenic plants which produce high levels of anthocyanins derived from delphinidin. In accordance with the present invention, gene constructs are generated and used to make transgenic plants which express high levels of delphinidin and/or its derivatives relative to non-transgenic plants of the same species. It has been determined in accordance with the present invention that genetic constructs which comprise a promoter from a gene encoding a flavonoid pathway enzyme operably linked to a flavonoid 3',5'-hydroxylase are capable of directing expression of high levels of delphinin-derived anthocyanins. The production of these high levels of delphinidin and related molecules is particularly useful in developing a range of plants exhibiting altered flower color properties.